Single-cell level LasR-mediated quorum sensing response of Pseudomonas aeruginosa to pulses of signal molecules

Quorum sensing (QS) is a communication form between bacteria via small signal molecules that enables global gene regulation as a function of cell density. We applied a microfluidic mother machine to study the kinetics of the QS response of Pseudomonas aeruginosa bacteria to additions and withdrawals of signal molecules. We traced the fast buildup and the subsequent considerably slower decay of a population-level and single-cell-level QS response. We applied a mathematical model to explain the results quantitatively. We found significant heterogeneity in QS on the single-cell level, which may result from variations in quorum-controlled gene expression and protein degradation. Heterogeneity correlates with cell lineage history, too. We used single-cell data to define and quantitatively characterize the population-level quorum state. We found that the population-level QS response is well-defined. The buildup of the quorum is fast upon signal molecule addition. At the same time, its decay is much slower following signal withdrawal, and the quorum may be maintained for several hours in the absence of the signal. Furthermore, the quorum sensing response of the population was largely repeatable in subsequent pulses of signal molecules.


Supplementary Methods
Well-plate assay to determine the lac promoter activity (in the PKRC12 plasmid) in the used P. aeruginosa PUPa3 strain P. aeruginosa PUPa3 DlasI and P. aeruginosa PUPa3 DlasR DrhlR (signal blind) strains were cultured overnight in LB (supplemented with 50 µg/ml kanamycin and gentamycin) until stationary phase in test tubes (30 °C, 200 rpm).The cultures were back-diluted the following morning by 1:1000 and further incubated until their optical density (measured at 600 nm) reached 0.15.Then, two different samples were prepared of each strain: 1) without 3O-C12-HSL, but 0.1 % ethyl acetate, 0.0001 % acetic acid were added; 2) with 1 µM 3O-C12-HSL, 0.1 % ethyl acetate, and 0.0001 % acetic acid.Six replicates were prepared for each sample and strain with a final volume of 100 µl per well.The experiment was carried out with a BioTek Synergy H1 microplate reader (Agilent Technologies, Inc., Santa Clara, CA USA) at 30 °C, using continuous shaking and reading the OD600 and fluorescence intensity (excitation 479 nm, emission 520 nm) values in every 5 minutes for 8 hours.LB (supplemented with 0.1 % ethyl acetate and 0.0001 % acetic acid) solution was used as reference and sterility control during the measurement.Fluorescence intensity data were normalized by the measured OD600 values (Supplementary Fig. S14).

Well-plate assay to determine GFP degradation
Three colonies of P. aeruginosa PUPa3 DlasI strain were grown overnight in LB (supplemented with 50 µg/ml kanamycin and gentamycin) until stationary phase in test tubes (30 °C, 200 rpm) and back-diluted in the following morning by 1:500.After 5 hours of incubation (OD600 reached 0.45-0.6;measured in the test tubes) 1 μM 3-oxo-C12-HSL was added into the media to induce QS, and incubation continued for 3 hours (during which their OD reached 1.15-1.40).The cultures were then centrifuged two times (5 min, 3500 rpm) and resuspended in PBS media (without signal molecules), in which no growth was expected.The optical density (at 600 nm) and fluorescence intensity of each PBS culture were monitored over time in a 96-well plate.Three replicates were prepared of each culture with 100 μl final volume per well.The experiment was carried out with a BioTek Synergy H1 microplate reader (Agilent Technologies, Inc., Santa Clara, CA USA) at 30 °C, using continuous shaking and reading the OD600 and fluorescence intensity (excitation 479 nm, emission 520 nm) values every 5 minutes for 2.5 hours.The measured OD did not change much, which indicates a constant cell number over this period (data not shown).Fluorescence data were fitted by exponentials to determine the time constant of the decay, which gives us information on the lifetime of GFP(ASV) in this strain (Supplementary Fig. S8).

Fluorescence microscopy study to determine the base intensity distribution of P. aeruginosa PUPa3
DlasI strain without QS stimulation on single-cell level A control experiment without QS stimulation of P. aeruginosa PUPa3 DlasI strain was performed using bacteria cultured in test tubes.Three colonies of P. aeruginosa PUPa3 DlasI strain were grown in LB (supplemented with 50 µg/ml kanamycin and gentamycin) until stationary phase (30 °C, 200 rpm), and back-diluted in the following morning by 1:1000.As the optical density (at 600 nm) reached 0.15, 0.1 % ethyl acetate and 0.0001 % acetic acid were added into each culture tube.The cell suspensions were further incubated in the shaker incubator for 24 hours.Then, brightfield and fluorescence images of single cells were taken.For this purpose, 1.5 µl volumes of each culture were dropped onto microscope glass slides and covered with coverslips.
A Nikon Eclipse Ti-E inverted microscope (Nikon Corp, Tokyo, Japan) equipped with a Prior Lumen 200 Pro excitation lamp (Prior Scientific Instruments Ltd, Cambridge, UK) set at 100 % intensity was used for imaging.A 40× Nikon Plan Fluor objective, a GFP fluorescence filter set (49002, Chroma Technology Corp., Bellows Falls, VT, United States), and a Prior Proscan II motorized stage (Prior Scientific Instruments Ltd, Cambridge, UK) were parts of the microscope setup.An Andor NEO sCMOS camera (Andor Technology Ltd, Belfast, UK) and NIS Elements Ar.Software (Nikon Corp, Tokyo, Japan) was used for image acquisition and microscope control.The following camera settings were used for fluorescence imaging: no binning, 100 ms exposure time, rolling shutter, 4 gain, and a bit depth of 11.
Microscopy images were analyzed using Fiji.Phase contrast images were thresholded and used to create a mask to find bacteria on the fluorescence images as well.The average fluorescence intensity of each cell was calculated, and a histogram was prepared to show the distribution of the average fluorescence intensity of single cells without the presence of the 3O-C12-HSL signal molecule (Supplementary Fig. S16).

Image analysis using the BACMMAN software
In the "Pre-processing" step in BACMMAN, background noise removal (Backgroundfit option with Sigma factor 1) and cropping to the region of interest were applied.The microchannels along the yaxis were positioned and aligned using the rotation and flip options.On the final pre-processed image, the microchannel dead-end was at the top of the image.
In the next "Processing" step (while pre-filtering options were omitted), microchannel segmentation/tracking of microchannels and bacteria was done.For microchannel segmentation, we used the MicrochannelFluo2D segmentation algorithm, where the first step was to define the length and width of the microchannel and the y-start shift.These were 154 px, 20-30 px, and 5-20 px, respectively.For thresholding, we used Backgroundfit with Sigma factor 1. The filling proportion parameter and the minimal object size were set to 0.05 and 3, respectively.
For the segmentation of bacterial cells, we used the BacteriaFluo segmentation algorithm, in which we chose the Hysteresis_thresholding as a foreground selection method.For background and foreground thresholding, we used Parent_track.The Method selected for these was Backgroundfit with Sigma factor 1 in both cases.
Two parameters affect the segmentation of dividing cells.For the Hessian scale, we used 2, and the Split Threshold was 0.001.Occasional errors in the BACMMAN image analysis (e.g., missensed divisions lasting for single frames) were manually checked and corrected.
An example of bacteria segmentation and tracking by BACMMAN is presented in Supplementary Fig. S15.

Background correction of the raw image data
A background correction was performed on the raw pixel-averaged fluorescence intensities (I) by applying the following formula: −   −  where B is the average background intensity (measured in the absence of fluorescent bacteria in the vicinity of the mother machine side channels) and D is a camera specific average dark intensity (measured with the camera sensor blocked from all light sources).

Measuring cell length and elongation rate
The population average of cell length and elongation rate was measured on fully aggregated datasets and on biological replicate-level (Supplementary Fig. S7).The parameter "spine length" calculated by the BACMANN software was used as cell length.This parameter is defined as the central line crossing the bacterium from one pole to the other.(Each point of the spine is equidistant from the two closest points of the contour located on each side of the spine.) Elongation rate is defined as the difference between cell length measured at the beginning and at the end of the cell cycle, divided by the cell cycle length.
Mathematical model to determine the QS threshold concentration By using different signal molecule concentrations (S) in the mathematical model of the QS response, the theoretical threshold was determined.The concentrations varied from 0 to 150 nM with 0.5 nM step size.Calculations were done using a lower and a higher static growth rate (calculated from the measured cell cycle length at the 6 h timepoint in the experiments with 10 nM (1.3 h) and 1 μM (4.3 h) signal concentration, respectively).The dynamics of the intensity change were calculated for 22 hours for each concentration.The maximum intensity values in the function of the corresponding concentrations are presented in Supplementary Fig. S10.The threshold concentration was determined by finding the midpoint of the curves, which was found to be in the 16.8-21.6nM range.

Sensitivity analysis of the model parameters
We performed a sensitivity analysis to determine the extent to which the change of one parameter affects the resulting kinetics.For this, we changed the fitted value of the model parameters by ±15 %, one at a time.The original fitted curve and the new curve (calculated using the changed parameter) were compared by calculating the ratio of the residual sum of squares and the total sum of squares (which is practically 1-R 2 ).This value is indicated in Supplementary Fig. S9.